Potato Fertility Restoration

ABSTRACT

A family 1 cellulose-binding-domain (CBD) encoding gene from  Phytophthora infestans  was used to develop transgenic Bintje potato plants. Tests with detached leaflets showed no evidence of increased or decreased resistance to  P. infestans , in comparison with the susceptible Bintje controls. Changes in plant morphology were most evident in the CBD1 multicopy transgenics. Plant height increases were evident in the later growth stages, along with earlier flowering and the ability to produce seed balls. While Bintje control plants are male and female sterile, the multicopy transgenics were male sterile and female fertile. Crosses made into Bintje demonstrated the ability to transfer  P. infestans  targeted R genes, as well as genes responsible for color and tuber shape, into Bintje germplasm. Selection for the absence of the CBD1 transgene should allow for immediate use of the material.

This application claims the benefit of U.S. Provisional Application No.62/042,504 filed Aug. 27, 2014, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the transformation of a sterile potatogenotype where the resulting transgenic potato plant exhibits changes inplant morphology and ovule development and produces fertile seedsallowing for successful potato breeding and resulting in new improvedcultivars. Removal of the transgene results in non-transgenic potatoplants and non-transgenic potatoes displaying new traits.

2. Description of the Relevant Art

Cellulose binding domains (CBDs) represent a subset of carbohydratebinding modules. CBDs are found associated with the majority ofsaprophyte-encoded cellulolytic enzymes; however, they are generally notfound associated with plant pathogen-encoded or with plant-encodedcellulolytic enzymes (Wang and Jones. 1995a. Appl. Environ. Microbiol.61:2004-2006; Wang and Jones. 1995b. Gene 158:125-128). The principlefunction of CBDs is to mediate adherence to the carbohydrate substrate(Boraston et al. 2004. Biochem. J. 382:769-781; Nerve et al. 2010. Proc.Natl. Acad. Sci. 107:15293-15298). An additional function can be foundin the ability of some CBDs to directly dissociate cellulosemicrofibrils (Lehtio et al. 2003. Proc. Natl. Acad. Sci. 100:484-488).Within the CBD families, there are a few examples of CBDs that arepresent as independent proteins, often associated with the cell wall. Anexample is CBD1 from the phytopathogen Phytophthora infestans (Jones andOspina-Giraldo. 2011. PLoS ONE 6(8):e23555). The P. infestans CBD1protein is found tightly associated with the cellulose-containing hyphalcell wall, and may play a role in assembly and/or integrity of the cellwall. This led to the idea that expression of this protein in potatoplants may alter the host:pathogen interactions through binding to thehost cellulose molecules, providing a potential shield from pathogenendoglucanases, or conversely, binding to Phytophthora hyphae, providinga shield from host endoglucanases.

Since the potato industry, like other parts of the agriculturalindustry, is facing many challenges, as for example, the problemsassociated with climate change, the spread of exotic pests andpathogens, a demand for reduced use of chemicals and a need forcommercially important desirable traits, new strategies for obtainingimproved varieties (cultivars) of potatoes are needed, particularly forthose potato varieties that are popular and widely used, but are alsosterile and thus not capable of being changed by conventional breedingpractices.

SUMMARY OF THE INVENTION

We have expressed the isolated CBD1 cDNA (SEQ ID NO:1) from Phytophthorainfestans in Bintje potato plants and confirmed that its expressionresults in changes in morphology and ovule development in thetransformed plants.

In accordance with this discovery, it is an object of the invention toprovide a strategy and model system to express the CBD1 gene in Bintjepotato plants and to use the strategy to obtain new traits in widelyused potato cultivars that are sterile.

It is an object of the invention to provide transformed Bintje potatoplants which grow larger wherein plant cells of said plants comprise arecombinant vector comprising the CBD1 gene.

It is another object of the invention to provide transformed Bintjepotato plants having changes in ovum development wherein said plantscomprise a recombinant vector comprising the CBD1 gene.

It is a further object of the invention to provide progeny resultingfrom crosses of CBD1 transgenic Bintje and pollen from other potatocultivars having advantageous traits, said progeny having advantageoustraits of Bintje together with other advantageous traits such as diseaseresistance and increased nutritional value, e. g. increased carotenoidlevels.

It is another object of the invention to provide a method of obtainingnew improved Bintje potato plants exhibiting new advantageous traitssuch as, for example, resistance to plant pathogens or increasednutritional value or yield comprising: transforming a regenerable tissueof a Solanum tuberosum Bintje cultivar plant with a vector comprising arecombinant construct comprising a P. infestans CBD1 cDNA and one ormore regulatory elements operatively linked to said cDNA wherein saidCBD1 cDNA encodes the polypeptide of SEQ ID NO:2 (GenBank AccessionNumber ABW76417.1); culturing the CBD1 transformed S. tuberosum Bintjeplant regenerable tissue in vitro; regenerating from said CBD1transformed S. tuberosum Bintje plant regenerable tissue CBD1 transgenicS. tuberosum Bintje plantlets; planting the CBD1 transgenic S. tuberosumBintje plantlets of uniform height in soil; maintaining said plantletsin a greenhouse; and selecting for a growing CBD1 transgenic S.tuberosum Bintje plant comprising said recombinant construct andexhibiting a greater growth rate as compared to other transgenics and aalso exhibiting change in ovum development with production of seedballs; fertilizing said plant with pollen from fertile potato plantsexhibiting said advantageous traits; and obtaining S. tuberosum BintjeCBD1 transgenic and non-transgenic plants which exhibit new advantageoustraits, whereas the parental S. tuberosum Bintje non-transgenic plant,being a sterile cultivar, could never have produced a S. tuberosumBintje potato plant with new advantageous traits.

It is yet another object of the invention to provide a method ofproducing Bintje potato plants with new advantageous traits such as, forexample, resistance to plant pathogens or increased nutritional value oryield comprising selecting those plants which exhibit the advantageoustrait but which do not comprise the CBD1 transgene, as the transgene isonly required for breeding.

It is a further object of the invention to provide seeds obtained fromfertilized Bintje transgenic plants expressing CBD1.

Other objects and advantages of this invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the U.S. Patent and TrademarkOffice upon request and payment of the necessary fee.

FIG. 1 depicts Southern blot analysis of CBD1 transgenic Bintje potatoplants. DNA was digested with Xba1, which is present at the 5′ end ofthe CBD1 gene.

FIG. 2 shows the growth rate for Bintje CBD1 transgenic and controlplants. Growth changes appear after 5 weeks of growth, with only certainlines exceeding the height of the control plants.

FIGS. 3A-3C depict floral and seed ball formation in Bintje CBD1transgenic plants. FIG. 3A shows earlier flowering; FIG. 3B depicts theabundant seed balls; and FIG. 3C shows that the largest seed balls wereproduced from CBD1 transgenic plant B-48.

FIGS. 4A-4C show leaves from a CBD1 transgenic Bintje B-48 plant, from aLenape E10 transgenic for the RB late blight resistance gene, and fromprogeny of the cross between CBD1 transgenic Bintje B-48 and Lenape E10transgenic for RB. FIG. 4A shows that the CBD1 transgenic Bintje B-48parental line is fully susceptible to P. infestans US 11. FIG. 4B showsthat the RB transgenic parental line is fully resistant. FIG. 4C showsthat a subset of progeny of the cross between CBD1 transgenic B-48 andLenape E10 transgenic for RB are fully resistant to late blight.

FIG. 5 shows the cross between CBD1 transgenic Bintje B-48 and cultivarPeter Wilcox. The range of tuber shapes and colors represent potentialfor development of Bintje.

DETAILED DESCRIPTION OF THE INVENTION

We have transformed Potato (Solanum tuberosum) Bintje with thePhytophthora infestans Cellulose Binding Domain 1 (CBD1) gene. Thisinvention concerns the first occurrence of induction of ovum developmentand seed balls in a sterile potato cultivar by expression of CBD1. Theovum development in S. tuberosum Bintje enables a previously sterilecultivar to overcome the limitations of sterility making possiblefertilization by pollen from plants carrying advantageous traits.

Potatoes are the fourth most important food crop in the world, resultingin billions of dollars in economic value. Surprisingly potato productionis highly dependent on two cultivars, Russet Burbank (US) and Bintje(Europe), each representing over 50% of total production. The cultivarBintje originated in 1910, yet it remains one of the most widely usedpotatoes in Europe. It has high yield, grows in many different soils andhas outstanding flavor. Unfortunately, some disadvantages are that itlacks disease resistance and that the tubers are short and consideredunsuitable for use commercially, for example, for French fries sold byfast food franchises. Use of the CBD1 gene in transgenic Bintje willallow for introduction of new traits into a previously infertilecultivar. In progeny from preliminary crosses, we have demonstrated thatresistance to the most important disease of potato (late blight causedby P. infestans) can be introduced into the highly susceptible Bintjeusing pollen from a potato line carrying the ARS patented Rb gene. Wehave demonstrated that skin color and carotenoid levels can be changedafter crosses with the purple skinned, yellow fleshed Peter Wilcoxpotato. Progeny are selected for absence of the CBD1 transgene,providing a null-segregant population that is considered non-transgenicand is therefore a product that does not require regulation. An improvednon-transgenic Bintje cultivar is critical to European development andwould accelerate its use in the US. We are currently developing CBD1transgenic Russet Burbank to determine if CBD1 can restore ovuledevelopment, as this popular cultivar is also considered sterile.

Potato breeding relies on crossing fertile pollen from one plant to thestigma of another plant. After successful fertilization, the ovule wallsexpand to produce a small seed ball, similar in appearance to a smallunripe tomato. At maturity the seed ball may have up to 200 seeds, whichare sown and screened for desired traits. Development of new potatovarieties can be limited by the infertility of germplasm, where normalflowers are produced, but no seed is produced as a result ofdeficiencies in the pollen and/or the ovules. The mechanism(s) of potatoinfertility has not been identified. During the screening of variousengineered potatoes, we noted that one transgene conferred the abilityof an infertile potato variety (Bintje) to produce seed balls (berries).Seed ball size was correlated with the transgene copy number in theindividual transformants. The original seed balls resulted from selfpollination but had no seed. Reciprocal crosses with pollen from otherpotatoes produced large numbers of viable seed in the balls after thesame pollination. The pollen from the transgenic Bintje lines failed tosupport seed production in other germplasm indicating that thetransgenic pollen remained sterile. Thus, the effect of the transgene ison development of the seed ball structure.

The CBD1 gene used in transgenic development was discovered in our lab.The encoded CBD1 protein (SEQ ID NO:2) functions as a cellulose bindingprotein. It was originally found in the walls of the potato pathogenPhytophthora infestans, which has cell walls composed of cellulosicglucans. There have been reports that cellulose binding proteins couldinfluence plant growth, and we were testing the possibility that P.infestans may use this protein to influence plant growth. Other reportshave found either growth stimulation or no effect after expressingcellulose binding proteins in plants. There are no reports on use of ourtransgene, or on the ability of any CBD1 to support ovule development.High copy number potato transformants produce the largest seed balls,but almost no tubers; therefore, we select away from the transgene aftersuccessful crosses are made, as the CBD1 is only needed for crossing.

We originally thought that introducing CBD1 into transgenic potato mightalter the host:pathogen interaction. One way this could happen would beif CBD1 acted in a manner similar to Cladosporium fulvum AVR4, where thechitin binding AVR4 protein protects the hyphae from plant chitinases,preventing release of elicitor fragments (Westerink et al. 2002. Mol.Plant Microb. Interact. 15:1219-1227; van den Burg et al. 2006. Mol.Plant Microb. Interact. 19:1420-1430). In a soybean: Phytophthora sojaeinteraction, the soybean produces glucanases that attack the cellulosicP. sojae hyphae, releasing elicitor fragments. CBD1 could be onemechanism for protecting the hyphae, along with the reported glucanaseinhibitor proteins (Bishop et al. 2005. Genetics 169:1009-1019). Such aninteraction would be expected to lead to greater host susceptibility,but this was not seen in our studies.

An alternative possibility would be that CBD1 interacts with the hostcell wall. Interactions with the host cell wall could increasesusceptibility if the CBD1 acted to loosen cellulose microfibrils, or,in contrast, could reduce susceptibility if the CBD1 bound to andshielded substrates susceptible to pathogen enzymes. While there was noevidence that these mechanisms were active, based on indistinguishabledifferences in susceptibility between control and transgenic CBD1plants, there was an obvious effect on gross plant morphology.

There is evidence to suggest that CBDs can interact with the plant cellwall. This is seen in the use of binding modules as molecular probes ofcell wall structure (Boraston et al., supra; Herve et al., supra). It isalso suggested in the limited reports of their use in transgenic plants.In one case, a family 3 CBD protein from the saprophytic bacteriumClostridium cellulovorans (Goldstein et al. 1993. J. Bacteriol.175:5762-5768), was found to increase early growth of transgenic Desireepotato plants (Shpigel et al. 1998. Plant Physiol. 117:1185-1194;Safra-Dassa et al. 2006. Mol. Breeding 17:355-364). At higher copynumbers there was an inhibition of growth. Interestingly, our use of afamily 1 CBD resulted in greater growth only at later stages of plantdevelopment, and higher copy number enhanced growth and ovuledevelopment. The use of a Family 22 xylan binding module in transgenictobacco failed to show any apparent effects on morphology or growthrates (Obembe, O. O. 2009. African J. Biotechnol. 8:6036-6039), whileanother study showed a marked reduction in growth of tobacco when atandem CBM was expressed in transgenic materials (Obembe et al. 2007. J.Plant Res. 120:605-617). Given the variable effects of CBDs intransgenic plants, there can be no assumptions made about the effect ofany carbohydrate binding modules until tested.

An unusual feature of the Phytophthora CBD 1 transgenic Bintje is theeffect on ovule formation. Even in the absence of fertilization, thetransgenic plants are able to produce seed balls. This would be similarto parthenocarpic fruit development, where fruit develops in the absenceof seed development. This has been engineered into Solanaceous cropsthrough manipulation of tissue-targeted auxin production (Rotino et al.1997. Nat. Biotechnol. 15:1398-1401). After successful fertilization,the Bintje seed balls are larger than those produced withoutfertilization, indicating a normal interaction between seed developmentand ovule growth. Auxin has a well known role in causing cell wallloosening and cell expansion through induction of various plant-encodedcarbohydrate modifying enzymes (Nishitani and Masuda. 1981. PhysiolPlantarum 52:482-494).

The mechanism of CBD1-mediated ovule development remains to becharacterized, however, CBD1 expression can be a useful tool fordeveloping the Bintje gene pool, one that hasn't changed since 1904(Stevenson, F. 1966. Amer. J. Potato Res. 43:458-459). The cultivarBintje is one of the most widely grown European cultivars (Retrievedfrom the Internet: europotato.org) due to strong yields, growth undervariable conditions, and excellent flavor after multiple differentcooking methods. Current limitations are lack of disease resistance andsmaller tuber size.

We demonstrate the ability to integrate single gene late blightresistance as well as color changes into the Bintje gene pool. Selectioncan be made for Bintje progeny that lack the CBD1 transgene, as itsprimary value is in breeding. This will allow for improved Bintjewithout carryover of transgenes, for those markets where that isdesired.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. 1987. Meth. Enzymol. 143:277) and particle-acceleratedor “gene gun” transformation technology (Klein et al. 1987. Nature(London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein byreference). Additional transformation methods are disclosed below. Thus,isolated polynucleotides of the present invention can be incorporatedinto recombinant constructs, typically DNA constructs, capable ofintroduction into and replication in a host cell. Such a construct canbe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. A number of vectors suitable for stabletransfection of plant cells or for the establishment of transgenicplants have been described in, e.g., Pouwels et al. 1985. Supp. 1987.Cloning Vectors: A Laboratory Manual; Weissbach and Weissbach. 1989.Methods for Plant Molecular Biology, Academic Press, New York; andFlevin et al. 1990. Plant Molecular Biology Manual, Kluwer AcademicPublishers, Boston. Typically, plant expression vectors include, forexample, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

As used herein, the terms “nucleic acid molecule”, “nucleic acidsequence”, “polynucleotide”, “polynucleotide sequence”, “nucleic acidfragment”, “isolated nucleic acid fragment” are used interchangeablyherein. These terms encompass nucleotide sequences and the like.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.However, isolated polynucleotides may contain polynucleotide sequenceswhich may have originally existed as extrachromosomal DNA but exist as anucleotide insertion within the isolated polynucleotide. Isolatedpolynucleotides may be purified from a host cell in which they naturallyoccur. Conventional nucleic acid purification methods known to skilledartisans may be used to obtain isolated polynucleotides. The term alsoembraces recombinant polynucleotides and chemically synthesizedpolynucleotides.

As used herein, “recombinant” refers to a nucleic acid molecule whichhas been obtained by manipulation of genetic material using restrictionenzymes, ligases, and similar genetic engineering techniques asdescribed by, for example, Sambrook et al. 1989. Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. or DNA Cloning: A Practical Approach, Vol. Iand II (Ed. D. N. Glover), IRL Press, Oxford, 1985.

A “construct” or “chimeric gene construct” refers to a nucleic acidsequence encoding a protein, here the CBD1 protein, operably linked to apromoter and/or other regulatory sequences.

As used herein, the term “express” or “expression” is defined to meantranscription alone. The regulatory elements are operably linked to thecoding sequence of the CBD1 gene such that the regulatory element iscapable of controlling expression of the CBD1 gene. “Altered levels” or“altered expression” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

As used herein, the terms “encoding”, “coding”, or “encoded” when usedin the context of a specified nucleic acid mean that the nucleic acidcomprises the requisite information to guide translation of thenucleotide sequence into a specified protein. The information by which aprotein is encoded is specified by the use of codons. A nucleic acidencoding a protein may comprise non-translated sequences (e.g., introns)within translated regions of the nucleic acid or may lack suchintervening non-translated sequences (e.g., as in cDNA).

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions. Thetissue-specificity of a promoter, for example, is exemplified by thepromoter sequence which specifically induces gene expression in roottips. Promoters that cause a nucleic acid fragment to be expressed inmost cell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg. 1989. Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be an RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to a DNA that is complementaryto and derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense” RNA refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense”, when used in the context of a particularnucleotide sequence, refers to the complementary strand of the referencetranscription product. “Antisense RNA” refers to an RNA transcript thatis complementary to all or part of a target primary transcript or mRNAand that blocks the expression of a target gene. The complementarity ofan antisense RNA may be with any part of the specific nucleotidesequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence,introns, or the coding sequence. “Functional RNA” refers to sense RNA,antisense RNA, ribozyme RNA, or other RNA that may not be translated butyet has an effect on cellular processes.

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the invention to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, stems, fruits,leaves, roots originating in transgenic plants or their progenypreviously transformed with a DNA molecule of the invention andtherefore consisting at least in part of transgenic cells, are also anobject of the present invention.

As used herein, the term “plant cell” includes, without limitation,seeds suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

The successful transformation of potatoes (Solanum) with CBD1 is a majorstep in overcoming sterility in popular potato varieties and will aid indevising new strategies for improving Solanum breeding thus ensuring thedevelopment of improved varieties of Solanum.

EXAMPLES

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein only to further illustrate the invention and are not intended tolimit the scope of the invention as defined by the claims.

Example 1 Plant Transformation and Selection

Transgenic Bintje was developed using 4 week old tissue culture-grownplantlets. Leaves were excised, cut laterally and immersed for 15 min inan acetosyringonone-induced culture of Agrobacterium tumefaciens LBA4404containing the CBD1 cDNA (SEQ ID NO: 1; GenBank Accession NumberEU179903.1) in the binary vector pBI121. Subsequent procedures wereessentially as reported previously (Banerjee et al. 2006. Plant Sci.170:732-738). Regenerated plants were maintained in a growth chamber onMurashige Skoog (MS) medium.

DNA samples from putative transformants were screened by PCR using 35Sspecific primers (35S-F gataatcatcgcaagaccggc [SEQ ID NO:3] and 35S-Rgacgtaagggatgacgcacaatccc [SEQ ID NO:4]) followed by sequencing of thePCR product. DNA from positive transformants were digested with Xba1,separated on a 0.7% agarose gel, transferred to nylon membrane andprobed with Dig-labeled CBD 1 DNA according to manufactures protocols(Roche).

Numerous transformants were obtained, and there were no apparentphenotypic changes seen during regeneration. Plantlets of uniform heightwere transferred to soil and maintained in a greenhouse. Measurementswere recorded weekly beginning at 5 weeks, when all plants wereestablished.

Copy numbers of CBD1 in the CBD1 transgenic plants varied (FIG. 1). CBD1transgenic plants B-4 and B-48 had higher copy numbers of CBD1. Thesetwo lines had greater growth rates relative to the other transgenics,while the other transgenics had growth rates similar to the controlplant (FIG. 2). Most of the growth increase occurred late in the growthcycle.

Example 2 Breeding Studies: RB Late Blight Resistance

During an initial round of transgenic plant assessment it was noticedthat the transgenic lines formed seed balls, unlike the control plantswhere the flowers abscised after blooming. The mature seed balls did notcontain matured seed. Two sets of reciprocal crosses were made usingBintje as the female parent. Pollen from a transgenic Lenape containingthe RB gene from Solanum bulbocastatum (Rommens et al. 2007. TrendsPlant Sci. 12:397-403) was applied to the stigma of Bintje transgenicsB-23 and B-48. Pollen from Bintje controls and the two transgenic lineswere applied to stigmas of the transgenic Lenape. Seed balls wereallowed to mature and seed harvested. A subset of seed from each of thesuccessful cross pollinations was planted to determine inheritance.

Crosses proved successful when CBD1 transgenic Bintje was the femaleparent. Transgenic Bintje remained male sterile due to the same lack ofpollen as found in the controls. Transgenic Bintje initiated floweringearlier, and each plant produced numerous seed balls, each with viableseeds, when out-crossed with donor pollen (FIG. 3). Crosses made tocontrol flowers with the same donor pollen, resulted in floralabscission, the usual phenotype.

To screen for late blight resistance, sporangia were harvested from twoweek old cultures of Phytophthora infestans (race US 11) by floodingplates with 5 ml sterile water and decanting the sporangia into asterile Petri dish. Harvested sporangia were refrigerated for one hour,followed by incubation at room temperature for 30 min to induce zoosporeformation. Leaflets from 6 week old greenhouse grown potato plants weredetached from the center of the plants and placed onto moistened papertowels in incubation trays. Fifty ml aliquots of the sporangia/zoosporemixture were applied at individual sites on the abaxial side of theleaves. Incubation trays were sealed with plastic wrap and placed in anincubator (18° C.). Inoculated leaves were kept in the dark for 24hours, followed by 14 hr light/10 dark lighting cycles. Disease progresswas scored relative to control Bintje leaflets.

There were no differences in the late blight susceptibility of CBD1transgenic Bintje in comparison with control plants, as all were highlysusceptible. The CBD1 transgenic Bintje B-48 parental line is fullysusceptible to P. infestans US 11 (FIG. 4A); the Lenape E10 transgenicfor RB is fully resistant (FIG. 4B). Single gene resistance to lateblight was successfully transmitted from a transgenic Lenape harboring acopy of the RB gene as evidenced by the fact that a subset of progeny ofthe cross between CBD1 transgenic B-48 and Lenape E10 transgenic for RBare fully resistant to late blight. Nine progeny were tested for lateblight resistance, four were susceptible like the CBD1-Bintje parent. Asubset of the progeny, five progeny, were, like the RB-Lenape parent,fully resistant to late blight (FIG. 4C).

Example 3 Breeding Studies: Color, Shape, Nutritive Value

Pollen from the purple skin, yellow fleshed Peter Wilcox were applied tothe stigma of Bintje transgenics B-23 and B-48. Pollen from Bintjecontrols and the two transgenic lines were applied to stigmas of PeterWilcox. Seed balls were allowed to mature and seed harvested. A subsetof seed from each of the successful cross pollinations was planted todetermine inheritance.

Crosses proved successful when CBD1 transgenic Bintje was the femaleparent. Transgenic Bintje remained male sterile due to the same lack ofpollen as found in the controls. Transgenic Bintje initiated floweringearlier, and each plant produced numerous seed balls, each with viableseeds, when out-crossed with donor pollen (FIG. 3). Crosses made tocontrol flowers with the same donor pollen, resulted in floralabscission, the usual phenotype.

Flavor is multi-gene dependent. Multigenic traits were successfullytransmitted after crossing the purple skin, yellow flesh Peter Wilcoxcultivar pollen to the CBD1 transgenic Bintje (FIG. 5). The resultingrange of tuber shapes and colors represent potential for successfuldevelopment of the parental flavorful CBD1 transgenic Bintje exhibitingnew advantageous traits. For example, the progeny of the crosses betweenCBD1 transgenic Bintje B-7-B-48 and Peter Wilcox exhibit varied tubershapes and have increased levels of carotenoids (FIG. 5).

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

The foregoing description and certain representative embodiments anddetails of the invention have been presented for purposes ofillustration and description of the invention. It is not intended to beexhaustive or to limit the invention to the precise forms disclosed. Itwill be apparent to practitioners skilled in this art that modificationsand variations may be made therein without departing from the scope ofthe invention.

We claim:
 1. A method of obtaining fertile female CBD1 transgenic Bintjepotato plants, said method comprising: a.) transforming a regenerabletissue of a Solanum tuberosum Bintje cultivar sterile plant with avector comprising a recombinant construct comprising a P. infestans CBD1cDNA and one or more regulatory elements operatively linked to said cDNAwherein said CBD1 cDNA encodes the polypeptide of SEQ ID NO:2; b.)culturing the CBD1 transformed S. tuberosum Bintje plant regenerabletissue in vitro; c.) regenerating from said CBD1 transformed S.tuberosum Bintje plant regenerable tissue CBD1 transgenic S. tuberosumBintje plantlets; d.) planting the CBD1 transgenic S. tuberosum Bintjeplantlets of uniform height in soil; e.) maintaining said plantlets in agreenhouse; and selecting for a growing CBD1 transgenic S. tuberosumBintje plant comprising said recombinant construct and exhibiting agreater growth rate as compared to other transgenics and also exhibitingchange in ovum development with production of seed balls, wherein saidgrowing CBD1 transgenic S. tuberosum Bintje plant is capable of beingfertilized by pollen from a male fertile S. tuberosum plant.
 2. Themethod of claim 1 wherein the CBD1 cDNA is identified by SEQ ID NO:1. 3.A transgenic S. tuberosum Bintje plant made by the method of claim 1, orprogeny thereof, wherein said plant or progeny thereof comprises theCBD1 recombinant construct and exhibits a change in ovum developmentwith production of seed balls and female fertility.
 4. A plant cell, aplant part or a plant tissue from the transgenic plant according toclaim 3, wherein the plant cell, plant part or plant tissue containsCBD1 cDNA.
 5. A transgenic seed of the transgenic plant according toclaim
 3. 6. A method of obtaining new improved Bintje potato plantsexhibiting new advantageous traits, said method comprising: a.)transforming a regenerable tissue of a Solanum tuberosum Bintje cultivarplant with a vector comprising a recombinant construct comprising a P.infestans CBD1 cDNA and one or more regulatory elements operativelylinked to said cDNA wherein said CBD1 cDNA encodes the polypeptide ofSEQ ID NO:2; b.) culturing the CBD1 transformed S. tuberosum Bintjeplant regenerable tissue in vitro; c.) regenerating from said CBD1transformed S. tuberosum Bintje plant regenerable tissue CBD1 transgenicS. tuberosum Bintje plantlets; d.) planting the CBD1 transgenic S.tuberosum Bintje plantlets of uniform height in soil; e.) maintainingsaid plantlets in a greenhouse; and selecting for a growing CBD1transgenic S. tuberosum Bintje plant comprising said recombinantconstruct and exhibiting a greater growth rate as compared to othertransgenics and a also exhibiting change in ovum development withproduction of seed balls; f.) fertilizing said plant with pollen frommale fertile potato plants exhibiting said advantageous traits; and g.)obtaining S. tuberosum Bintje CBD1 transgenic and non-transgenic plantswhich exhibit new advantageous traits, wherein the parental S. tuberosumBintje non-transgenic plant, being a sterile cultivar, was not capableof producing a S. tuberosum Bintje potato plant with new advantageoustraits.
 7. The method of claim 6 wherein the CBD1 cDNA is identified bySEQ ID NO:1.
 8. A S. tuberosum Bintje plant made by the method of claim6, or progeny thereof, wherein said plant or progeny thereof is a S.tuberosum Bintje plant which exhibits new advantageous traits.
 9. Theplant of claim 8 wherein the advantageous trait is any heritable trait.10. The plant of claim 9 wherein the advantageous heritable trait is anyone of resistance to plant pathogens, increased nutritional value,increased yield and color.
 11. The plant of claim 8 wherein the saidplant is a non-transgenic plant.
 12. A plant cell, a plant part, a planttissue or a plant seed of the plant of claim
 11. 13. The plant of claim8 wherein the said plant is a transgenic plant.
 14. A plant cell, aplant part, a plant tissue or a plant seed of the plant of claim 13.